Project Summary According to the World Health Organization, approximately 36 million people worldwide were living with HIV-1 at the end of 2015 and 1.1 million people died from this disease during the same year. Vaccination is the most effective strategy to prevent infectious diseases, and successful vaccines are usually protective because they elicit antibodies that neutralize the pathogen (1). Although there is no protective vaccine against HIV-1, broadly neutralizing antibodies (bNAbs) isolated from infected patients are protective in animal models of infection even at relatively low concentrations. These antibodies are potent neutralizers that recognize conserved features of the virus envelope spike (Env) that are shared among diverse strains of the virus, and it is generally agreed that a vaccine that elicits bNAbs would be protective against HIV-1 infection. However, with the exception of llamas and genetically engineered mice, bNAbs have not been elicited by vaccination (2). The experiments in genetically engineered knock-in mice showed that bNAb development required germline-targeting Env-antigens that were specifically designed to activate B cells expressing the germline precursor antibodies that correspond to bNAbs (3-5). In addition, singular antigens were not sufficient, and bNAb development required a sequence of specific immunogens delivered in order (5-8). However, the immunization schemes devised in knock-in mice could not be extended to wild type (wt) mice or other animals in part because of lack of understanding of the relationship between germline antigen affinity and bNAb precursor B cell frequency in initiating a productive immune response in the presence of competing polyclonal B cells. The objects of the proposed research are to: 1. define the precise relationship between precursor B cell frequency and affinity to cognate antigen, in recruiting bNAb precursors into the germinal center; 2. test the idea that pre-expansion of specific precursors using anti-idiotypic antibodies will facilitate the development of anti-CD4bs antibodies. The relationship between affinity and precursor B cell frequency will be defined in adoptive transfer experiments using antigens provided by Drs. Stamatatos and McGuire. New vaccination approaches using anti-idiotypic antibodies to expand bNAb precursor frequency before vaccination will be tested in three different mouse models with variable levels of polyclonality: I) In HC only knock-in mice which have the lowest level of polyclonality due to variable mouse light chains II) adoptive transfer of variable numbers of knock-in B cells expressing a single Env-specific BCR into wt mice and III) fully polyclonal mice expressing human germline Ig genes. The information obtained by these experiments will advance our understanding of how to approach the problem of how to develop a protective vaccine against HIV-1.